Enantiopure haptens for nicotine vaccine development

ABSTRACT

The invention provides chiral haptens (−)-3′-AmNic and (−)-N4N for use in generating antibodies in a patient specific for (−)-nicotine. The haptens can be conjugated to suitable carrier proteins and administered as an antigenic mixture, optionally comprising adjuvant(s), to a patient suffering from nicotine or tobacco addiction or habituation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject patent application is a continuation of U.S. patent application Ser. No. 15/311,322 (filed Nov. 15, 2016); now pending), which is a § 371 U.S. National Phase filing of PCT/US2015/031583 (filed May 19, 2015; now expired), which claims the benefit of priority to U.S. Provisional Patent Application No. 62/000,312 (filed May 19, 2014; now abandoned). The full disclosures of the priority applications are incorporated herein by reference in their entirety and for all purposes.

BACKGROUND

According to the World Health Organization, there are over 1 billion smokers worldwide, and smoking is responsible for nearly 6 million deaths annually.(1) The economic impact is also sobering: in the United States alone, smoking costs nearly $300 billion in medical expenses and lost productivity each year.(2) The epidemiological link between chronic tobacco use and myriad diseases is well understood, and while many smokers wish to quit, currently available cessation aids do not help much. Synthetic small molecule agonists or antagonists target brain receptors implicated in nicotine dependence.(3-5) Acting centrally, these medicines produce an array of side effects.(6)

Meanwhile, we have been pursuing a pharmacokinetic (antibody-based) instead of a pharmacokinetic (drug-based) strategy to aiding smokers' efforts to quit.(7) Nicotine plays a central role in precipitating addiction to smoking tobacco. A nicotine vaccine stimulates the immune system to identify nicotine as a foreign antigen, eliciting antibodies that alter nicotine pharmacokinetics. Anti-nicotine antibodies reduce the concentration of free nicotine in the blood and prevent it from entering the central nervous system. Blocking the activation of brain reward systems can facilitate extinction of the addictive behavior, leading to better smoking cessation outcomes. A clinically approved nicotine vaccine would be a complementary addition to the available tools, which, when leveraged appropriately, could afford significantly better rates of sustained smoking abstinence.

NicVAX® represents the most clinically advanced nicotine vaccine to date, having progressed all the way through Phase III.(8-11) It was safe and well tolerated, but was effective for only a fraction of clinical trial participants.(12, 13) Nevertheless, given the huge promise of a clinically approved nicotine vaccine, research continues unmitigated. Many design and formulation aspects have been scrutinized in recent years to furnish something better then NicVAX®. Efforts include boosting immunogenicity through the use of newer adjuvants(14-17), improving practicality through alternative routes of administration(18), and adopting multivalent strategies(19-22) to increase anti-nicotine antibody binding capacity.

For a vaccine aimed at conferring protective immunity against a specific small molecule such as nicotine, it is important that the vaccine possess adequate chemical epitope homogeneity.(23-25) Other vaccines may be engineered to simultaneously target multiple prevailing epitopes, as in the case of diphtheria-tetanus-acellular pertussis (DTaP), measles-mumps-rubella (MMR), and 23-valent pneumococcal combination vaccines.(26, 27)

SUMMARY

The invention is directed, in various embodiments, to haptens suitable for use in raising antibodies to (−)-nicotine; to antigens comprising the haptens; to antibodies prepared using the antigens comprising the haptens; to vaccines comprising the anti-nicotine antigens; and to methods of treatment of tobacco habituation or addiction in patients comprising the use of anti-nicotine vaccines/antigens.

The haptens, in various embodiments, have sufficient structural similarity to the alkaloid (−)-nicotine, the major drug component of tobacco (Nicotiana), such that antibodies raised, e.g., in a human patient, against antigens comprising the haptens of the invention also react with (−)-nicotine, and thus the antigens can be suitable for administration to patients for raising anti-nicotine antibodies by means of the patient's immune system. Such antibodies can serve to bind nicotine, such as from smoked or chewed forms of tobacco, and block the drug effect on the patient that serves to reinforce the drug addiction in the patient. Furthermore, the antibodies generated by use of the antigenic vaccines of the invention can be selective for (−)-nicotine (i.e., (S)-nicotine).

In various embodiments, the invention provides a hapten of formula (−)-3′-AmNic

a conjugated hapten comprising (−)-3′-AmNic wherein the (−)-3′-AmNic hapten is covalently bonded via a linker to a carrier protein; an antigen for administration to a patient, comprising the conjugated hapten and optionally adjuvant(s); an antiserum produced in a patient comprising antibodies having immunological affinity for (−)-nicotine; and a method of treatment of nicotine or tobacco addiction or habituation comprising administering an effective amount of the conjugated hapten or an antigenic mixture comprising the conjugated hapten to a patient suffering from the addiction or habituation, such that antibodies are produced in the patient having affinity for (−)-nicotine.

In various embodiments, the invention provides a (−)-nicotine hapten of formula (−)-N4N

a conjugated hapten comprising (−)-N4N wherein the (−)-N4N hapten is covalently bonded via a linker to a carrier protein; an antigen for administration to a patient, comprising the conjugated hapten and optionally adjuvant(s); an antiserum produced in a patient comprising antibodies having immunological affinity for (−)-nicotine; and a method of treatment of nicotine or tobacco addiction or habituation comprising administering an effective amount of the conjugated hapten or an antigenic mixture comprising the conjugated hapten to a patient suffering from the addiction or habituation, such that antibodies are produced in the patient having affinity for (−)-nicotine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a synthetic protocol for the preparation of (−)-3′-AmNic and (−)-3′-AmNic, which were then conjugated via a succinate linker to tetanus toxin (“TT”) and evaluated for their specificity towards (−)-nicotine.

FIG. 2 shows a synthetic protocol for the preparation and conjugation of (−)-N4N.

FIG. 3 depicts relative ELISA titers for antisera from vaccine groups, measured against two coating antigens. Each value depicted by a symbol is a mean (n=7). Mean titer for (−)-N4N-SucFliC antisera against (−)-N4N-SucBSA coating antigen was normalized to 1.0, and all other data points represent mean ELISA titers, relative to this antisera/coating antigen combination.

FIG. 4 shows binding affinity curves for radioimmunoassays (RIA) (−)-3′-AmNic and (+)-3′-AmNic.

FIG. 5 is a graph summarizing results from an antinociception assay of four nicotine vaccines relative to saline control in mice. Each bar depicted is a mean (n=7) with error bar as SEM. Ordinary one-way ANOVA with uncorrected Fisher's LSD, * P<0.05 for (−)-N4N-SucFliC vs. Saline.

FIG. 6 summarizes results from a hypothermia assay for measuring efficacy of four nicotine vaccines in attenuating the effect of nicotine in mice. Each bar depicted is a mean (n=7) with error bar as SEM. Two-way RM ANOVA with Dunnett's multiple comparisons test, * P<0.05 for annotated vaccine group vs. Saline, ** P<0.005 for annotated vaccine group vs. Saline, *** P<0.0001 for annotated vaccine group vs. Saline.

DETAILED DESCRIPTION

The inventive hapten-carrier conjugates and antigen compositions specifically and selectively target only (−)-nicotine; thus, in accordance with the invention, a vaccine according to embodiments described herein efficiently elicit antibodies capable of sequestering only (−)-nicotine. The notion that antibodies can enantiodifferentiate was first appreciated by Landsteiner nearly a century ago(28, 29) and continues to be exploited to this day. Such work includes enantiosclective catalytic antibodies (30-32) and stereospecific mAb to nicotine(33) and cocaine.(34-36) In the case of the nicotine mAb study, hybridomas were selected using (S)-(−)-[3H]nicotine, thereby optimizing for antibodies specific for the naturally occurring isomer.(33) The present invention exploits a capacity of antibodies to enantiodifferentiatc in developing vaccines for nicotine.

Many of the nicotine vaccines that have undergone clinical evaluation began with manipulation of racemic trans-cotinine carboxylic acid ((±)-1, FIG. 1). However, no chiral separation step (nor asymmetric synthesis step) was included in the production of the hapten-protein conjugate that would become NicVAX®. The present invention surprisingly shows that a non-racemic, fully (−)-nicotine vaccine conjugate is a superior immunogen, owing to the exquisite ability of antibodies to stereodifferentiate.

Haptens

The term “hapten” as used in the present invention is a low-molecular weight organic compound that, by itself, is incapable of eliciting an immune response. However, it will elicit an immune response once attached to a carrier molecule. According to some embodiments, the hapten is attached to the carrier via a linker.

According to one embodiment, a hapten of the present invention is a nicotine derivative that is trans-3′-aminomethylnicotine (3′AmNic). In accordance with one embodiment, for example, the hapten is a single enantiomer of trans-3′-aminomethylnicotine, specifically (−)-3′-AmNic. Both enantiomers are shown below:

In other embodiments, the hapten is (S)-3-(3-(1-Methylpyrrolidin-2-yl)pyridin-4-yl)propan-1-amine, (−)-N4N, as shown below:

By locating the linker attachment at the C4-position on the pyridyl ring of nicotine, no new sp³ stereocenters are introduced, in contrast to the linker attachment at the 3′-position on the pyrrolidine ring in either 3′-AmNic hapten shown above. Hence, no additional stereochemical complexity is thus introduced. A further advantage of (−)-N4N arises from the use of (−)-nicotine as the starting material for the synthesis of(−)-N4N, and by performing synthetic transformations that maintain enantiopurity throughout the chemical synthesis of this nicotine hapten: the need for a chiral separation step is obviated (FIG. 2). In addition, the nature of the linker itself, methylene (CH₂ attached to C4-position) is superior because it does not electronically perturb nicotine's pyridyl ring the way that a heteroatom (e.g., oxygen) does at this position. Thus, both steric and stereoelectronic considerations are made, with the goal of eliciting a better anti-nicotine antibody response (higher titers or concentrations, higher affinity and specificity, superior functional antagonism of nicotine's pharmacokinetics and pharmacodynamics) by virtue of this novel nicotine hapten design.

Conjugates

According to some embodiments, a nicotine hapten is directly attached to a carrier with or without a linker. For example, a single nicotine hapten can be attached to each available amine group on a carrier protein. General methods for directly conjugating haptens to carrier proteins, using a homobifunctional or a heterobifunctional cross-linker are well known in the art, for example, by G. T. Hermanson in Bioconjugate Techniques, Academic Press (1996) and Dick and Beurret in Conjugate Vaccines. Contribu. Microbiol. Immunol., Karger, Basal (1989) vol. 10, 48-114.

Direct conjugation using bifunctional crosslinkers generally results in a molar ratio of hapten to protein being limited by the number of functional groups available on the protein for the specific conjugation chemistry. For example, a carrier protein possessing n number of lysine moieties theoretically presents n+1 primary amines (including the terminal amino) available for reaction with a linker carboxyl group. Thus, direct conjugation gives rise to formation of n+1 amido bonds, i.e., a maximum of n+1 haptens attached. The skilled person will recognize that conjugated hapten density can depend upon concentration of the reactants used to conjugate the nicotine hapten to the carrier protein, and the nature of the carrier protein. Also, within a given preparation of nicotine-carrier conjugate, there will be variation in the hapten/carrier ratio of each individual conjugate. According to some embodiments, hapten density (molar ratio of conjugated hapten to protein carrier) can range from about 10 to about 70, from about 20 to about 60, and from about 30 to about 50.

Carrier Proteins

Once the nicotine hapten is prepared, according to any of the embodiments herein described, the haptent is then conjugated to a carrier protein which will be used to raise antibodies to the nicotine carrier conjugate. In some embodiments, the carrier protein of the present invention generally is any suitable immunogenic protein or polypeptide. An “immunogenic” molecule is one that is capable of eliciting an immune response. For instance, in one embodiment, the carrier protein is a T-cell epitope.

In other embodiments, the “carrier protein” is a multi-antigenic peptide (MAP), which is a branched peptide. By using a MAP, hapten density and valency are maximized because of multiple branched amino acid residues. Examples of amino acids that can be used to form a MAP include, but are not limited to, lysine.

In some embodiments, a carrier protein comprises a molecule containing at least one T cell epitope which is capable of stimulating the T cells of the subject, which subsequently induces B cells to produce antibodies against the entire hapten-carrier conjugate molecule. The term “epitope” as used herein includes any determinant on an antigen that is responsible for its specific interaction with an antibody molecule. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

In accordance with these embodiments and others, a carrier protein is selected based upon its ability to elicit a strong immunogenic response so that a diverse population of patients can be treated by the inventive hapten-carrier conjugates. For example, the carrier protein must be sufficiently foreign to elicit a strong immune response to the vaccine. Typically, the carrier protein in this regard is a large molecule capable of imparting immunogenicity to a covalently-linked hapten.

Many proteins known to the person skilled in conjugate vaccines are suitable for use in the present invention. For instance, in some embodiments the carrier is one that is used in the preparation of therapeutic conjugate vaccines, such as a number of toxins of pathogenic bacteria and their toxoids. Examples include diphtheria and tetanus toxins and their medically acceptable corresponding toxoids. In other embodiments, the carrier is bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), both of which are commonly used as carriers in the development of conjugate vaccines when experimenting with animals. In still other embodiments, the protein is flagellar filament structural protein (FliC).

In accordance with still other embodiments, the carrier is a protein that is antigenically similar to bacterial toxins, often referred to as a cross-reacting materials (CRM).

Conjugation of Hapten to Carrier Protein

In accordance with some embodiments, nicotine hapten-carrier conjugates of the present invention are prepared by reacting one or more haptens with a carrier protein to yield a hapten carrier conjugate.

A variety of functional groups are used to facilitate the linking or conjugation of a carrier to a hapten of the present invention. These include functional moieties such as carboxylic acids, anhydrides, mixed anhydrides, acyl halides, acyl azides, alkyl halides, N-maleimides, imino esters, isocyanates, amines, thiols, and isothiocyanates and others that are capable of forming a covalent bond with a reactive group of a protein molecule. Depending upon the functional moiety used, according to some embodiments, the reactive group is the amino group of a lysine residue or a thiol group on a carrier protein or a modified carrier protein molecule which, when reacted, results in amide, amine, thioether, amidine urea or thiourea bond formation. Other suitable activating groups and conjugation techniques are well known in the art (Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Inc. (1991); Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press: 1996; and Dick and Beurret in Conjugate Vaccines. Contribu. Microbiol. Immunol., Karger, Basal (1989) vol. 10, 48-114.)

In some embodiments, the linker is a linear moiety for conjugation of haptens to carrier proteins. For example, the linker is a succinyl moiety. Another example of a linker is adipic acid dihydrazide (ADH).

Antiserum and Antibodies

The antiserum of the present invention, according to some embodiments, comprises antibodies that are produced in response to an antigen, which itself comprises a hapten-carrier conjugate as described herein. In this context, techniques for making monoclonal antibodies are well-known in the art. For instance, monoclonal antibodies can be obtained by injecting mice with a composition comprising the nicotine hapten-carrier conjugate, subsequently verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the hapten-carrier conjugate, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

Alternatively, monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. The techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Techniques for preparing polyclonal antibodies also are well-known in the art. In general, for instance, an animal is injected with immunogenic material and then antibody rich serum is collected which contains therein a mixture of antibodies that are directed against numerous epitopes of the immunogen that was injected. Suitable host mammals for the production of antibodies include, but are not limited to, humans, rats, mice, rabbits, and goats.

In accordance with some embodiments of the present invention, functional antibody fragments also can be utilized. The fragments are produced by methods that include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction.

Alternatively, antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer such as those supplied commercially by Applied Biosystems, Multiple Peptide Systems and others, or they may be produced manually, using techniques well known in the art (Geysen et al., J. Immunol. Methods 102: 259 (1978)). Direct determination of the amino acid sequences of the variable regions of the heavy and light chains of the monoclonal antibodies according to the invention can be carried out using conventional techniques.

A fragment according to some embodiments of the present invention is an Fv fragment. An Fv fragment of an antibody is made up of the variable region of the heavy chain (Vh) of an antibody and the variable region of the light chain of an antibody (VI). Proteolytic cleavage of an antibody can produce double chain Fv fragments in which the Vh and VI regions remain non-covalently associated and retain antigen binding capacity. Fv fragments also include recombinant single chain antibody molecules in which the light and heavy chain variable regions are connected by a peptide linker (Skerra, et al. Science, 240, 1038-41 (1988)). Antibody fragments according to other embodiments of invention include Fab, Fab′, F(ab)₂, and F(ab′)₂, which lack the Fc fragment of an intact antibody.

Therapeutic Methods

Because nicotine exerts many of its significant effects after it crosses the blood brain barrier, the present invention provides therapeutic methods and uses for preventing nicotine from crossing the blood brain barrier. In particular, administration of a nicotine hapten-carrier conjugate to a patient generates antibodies against nicotine in the bloodstream of the patient.

Alternatively, anti-nicotine antibodies generated in a suitable host mammal and outside the body of the patient to be treated can be administered to a patient. If the patient smokes, the nicotine in his blood will be bound by the circulating anti-nicotine antibodies, preventing the nicotine from reaching the brain. Therefore, the antibodies prevent the physiological and psychological effects of nicotine that originate in the brain. Because the smoker will experience a lessening or cessation of these effects, he/she will lose the desire to smoke. The same therapeutic effects result if a patient uses smokeless tobacco, after being immunized with a nicotine hapten-carrier conjugate of the invention. Additionally, the conjugates and antibodies of the invention exert their effects by affecting the ability of nicotine to stimulate the peripheral nervous system.

Administration of Hapten-Carrier Conjugates

The conjugates of the invention are suitable for treating and preventing nicotine addiction. For treating nicotine addiction, a nicotine-carrier conjugate of the invention is administered to a patient suffering from nicotine addiction. For preventing nicotine addiction, patients at risk for developing nicotine addiction, such as teenagers, are treated with a conjugate according to the invention. Direct administration of the conjugate to a patient is called “active immunization.”

A vaccine composition of the present invention comprises at least one nicotine hapten-carrier conjugate in an amount sufficient to elicit an immune response thereto. The nicotine hapten carrier conjugate is capable of remaining in vivo at a concentration sufficient to be active against subsequent intake of nicotine.

Initial vaccination with the nicotine hapten carrier conjugate of the present invention creates high titers of antibodies that are specific to nicotine. The therapeutically effective amount of a conjugate which is administered to a patient in need of treatment for nicotine addiction is readily determined by the skilled artisan. Suitable dosage ranges are 1-1000 μg/dose. It generally takes a patient one to several weeks to generate antibodies against a foreign antigen. The production of antibodies in a patient's blood can be monitored by using techniques that are well-known to the skilled artisan, such as ELISA, radioimmunoassay (RIA), and Western blotting methods. Therapeutic effectiveness also can be monitored by assessing various physical effects of nicotine, such as blood pressure.

As described in detail below, the inventive nicotine hapten-carrier conjugates can be processed to afford a composition that is administered to a patient. According to some embodiments, modes of administration include but are not limited to intranasal, intratracheal, oral, dermal, transmucosal subcutaneous injection and intravenous injection. The skilled artisan will recognize that the initial injection may be followed by subsequent administration of one or more “boosters” of conjugate. The booster increases the production of antibodies against the nicotine hapten-carrier conjugate of the invention.

In some embodiments, the vaccine or antiserum compositions of the present invention comprises at least one adjuvant. The adjuvant is selected so that the effect of the carrier protein is not inhibited. Adjuvants those which are physiologically acceptable to humans; these include, but are not limited to, alum, QS-21, saponin and MPLA (monophosphoryl lipid A).

The vaccine compositions according to other embodiments optionally comprise one or more pharmaceutically acceptable excipients. For instance, the excipients include one or more of sterile water, salt solutions such as saline, sodium phosphate, sodium chloride, alcohol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycol, gelatin, mannitol, carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters, hydroxy methyl cellulose and buffers. Any additional excipients known to the skilled artisan are useful in the present invention.

The hapten-carrier conjugates of the present invention are incorporated into a pharmaceutical composition for administering to a patient in need of treatment or prevention of nicotine addiction. When the composition containing the hapten-carrier conjugate is to be used for injection, for instance, the hapten-carrier conjugate is solubilized in an aqueous, saline solution at a pharmaceutically acceptable pH. However, it is possible to use an injectable suspension of the hapten-carrier conjugate. In addition to the usual pharmaceutically acceptable excipients, the composition can contain optional components to ensure purity, enhance bioavailability and/or increase penetration.

In some embodiments, the vaccine composition optionally contains at least one auxiliary agent, such as dispersion media, coatings, microspheres, liposomes, microcapsules, lipids, surfactants, lubricants, preservatives and stabilizers. Any additional auxiliary agents known to the skilled artisan are useful in the present invention. Also useful herein are any agents which act to synergize the effect of the present vaccine composition.

The pharmaceutical composition of the present invention is sterile and is sufficiently stable to withstand storage, distribution, and use. Additionally, the composition may contain additional components in order to protect the composition from infestation with, and growth of, microorganisms. For example, the composition is manufactured in the form of a lyophilized powder that is reconstituted by a pharmaceutically acceptable diluent just prior to administration. Methods of preparing sterile injectable solutions are well known to the skilled artisan and include, but are not limited to, vacuum drying, freeze-drying, and spin drying. These techniques yield a powder of the active ingredient along with any additional excipient incorporated into the pre-mix.

Administration of Antibodies

Passive immunization comprises administration of or exposure to a polyclonal antibody or monoclonal antibody which has been raised in response to a nicotine hapten carrier conjugate of the invention. Such antibodies can be generated in animals or humans. Antibodies raised in response to a nicotine conjugate of the invention can be administered to prevent addiction to nicotine. For example, such antibodies can be administered to people considered to be at risk for developing addiction to nicotine, such as teenagers. Antibodies also are suitable for treating a patient addicted to nicotine. As discussed above, the antibodies bind nicotine in the blood, and prevent nicotine from crossing the blood brain barrier. According to some embodiments, antibodies raised by administration of the inventive hapten-carrier conjugate have a molecular weight range of from about 150 kDa to about 1,000 kDa.

The therapeutically effective amount of a therapeutic antibody of the invention which is administered to a patient in need of treatment for nicotine addiction is readily determined by the skilled artisan. Suitable dosage ranges are 1-1000 μg/dose.

A therapeutic composition according to some embodiments of the present invention comprises at least one antibody produced in response to a nicotine-carrier conjugate of the invention. The compositions optionally contain one or more pharmaceutically acceptable excipients. Useful excipients include sterile water, salt solutions such as saline, sodium phosphate, sodium chloride, alcohol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycol, gelatin, mannitol, carbohydrates, magnesium stearate, viscous paraffin, fatty acid esters, hydroxy methyl cellulose and buffers. Any additional excipients known to the skilled artisan are useful in the present invention.

The antibodies of the present invention, in order to be administered to a patient in need of treatment or prevention of nicotine addiction, are incorporated into a pharmaceutical composition. The composition comprising an antibody can be formulated in an aqueous, saline solution at a pharmaceutically acceptable pH for injection. However, it is possible to use an injectable suspension of the antibody. In addition to the usual pharmaceutically acceptable excipients, the composition contains optional components to ensure purity, enhance bioavailability and/or increase penetration.

A pharmaceutical composition comprising an antibody of the present invention is sterile and is sufficiently stable to withstand storage, distribution, and use. Additionally, the composition optionally contains additional components in order to protect the composition from infestation with, and growth of, microorganisms. Methods of preparing sterile injectable solutions are well known to the skilled artisan and include, but are not limited to, vacuum drying, freeze-drying, and spin drying. These techniques yield a powder of the active ingredient along with any additional excipient incorporated into the pre-mix.

EXAMPLES

The following examples constitute additional embodiments of the invention. The examples are therefore intended to illustrate, but not limit in any way, the invention described herein.

General Procedures Chemistry.

All reactions were carried out under an argon atmosphere with dry solvents using anhydrous conditions unless otherwise stated. Most chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) and used as received. Flagellin protein (FIiC) was prepared in-house. Tetanus toxoid (TT) was purchased from Statens Serum lnstitut (Copenhagen, Denmark). Yields refer to chromatographically (HPLC) and spectroscopically (¹H NMR) homogeneous (≧95%) materials. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV light as the visualizing agent. Flash column chromatography was performed using E. Merck silica gel (60, particle size 0.040-0.063 mm). Organic solvents were concentrated on a rotary evaporator under reduced pressure, followed by further evacuation using a dual stage mechanical pump. NMR spectra were recorded on a Bruker Avance III HD with DCH CryoProbe (600 MHz) instrument or a Bruker BioSpin DRX (500 MHz) instrument and calibrated using residual undeuterated solvent as an internal reference (CD₃OD @ δ 4.87 ppm ¹H NMR, δ 49.00 ppm ¹³C NMR). The following abbreviations (or combinations thereof) are used to explain ¹H NMR multiplicities: s=singlet, d=doublet, t=triplet, m=multiplet. High-resolution mass spectra (HRMS) were recorded on an Agilent LC/MSD TOF mass spectrometer by electrospray ionization time-of-flight reflectron experiments. JR spectra were recorded on a Thermo Scientific Nicolet 380 FTIR spectrometer.

Biology.

Each hapten-protein conjugate was mixed with phosphorothioated cytosine-phosphorothioate-guaninc oligodeoxynucleotide (“CpG ODN”) 1826 (Eurofins MWG Operon) and diluted to 1.0 mg/mL in pH 7.4 PBS. Then, an equal volume of Alhydrogel 2% (vac-alu-50, InvivoGen) was added dropwise, followed by 10 min of gentle inversion. Vaccines prepared in this manner contained 50 jig of conjugate, 50 μg of CpG, and 20 μL of Alhydrogel per 100 μL of complete formulation.

Example 1: Synthesis of (−)-3′-AmNic

First, racemic trans-3′-aminomcthylnicotine (3′-AmNic, (±)-2) was prepared from commercially available racemic trans-cotininecarboxylic acid ((−)-1). Next, using chiral supercritical fluid chromatography (SFC), ˜600 mg of (±)-2 was separated into ˜250 mg of each enantiomer (FIG. 1).

Example 2: (−)-3′-AmNic Tetanus Toxoid Conjugate and Vaccine

A. Conjugate.

Given our prior experience (37-39) coupling carboxylate-containing nicotine haptens to carrier proteins, we tried to do the same in the present context. For this, each enantiomer of 2 prepared according to Example 1 was acylated with succinic anhydride. However, activation of succinylated haptens and mixing with tetanus toxoid (“TT”) gave conjugates with low hapten densities.

Therefore, we employed an alternative choreography (Scheme 1, inset), in which the carrier protein (rather than the hapten) was first succinylated.(17, 40, 41) Hence, TT was treated with succinic anhydride in pH 8.65 Tris buffer to give SucTT. Then, SucTT was treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and either (−)- or (+)-2 in pH 5.80 2-(N-morpholino)ethane-sulfonic acid (“MES”) buffer, with final dialysis against pH 7.4 PBS. This procedure yielded separate quantities of (−)- and (+)-3′-AmNicSucTT (hapten densities >40 by MALDI-TOF analysis) suitable for formulation with adjuvants as described below.

B. Vaccine.

Each of the two hapten-protein conjugates as described above was mixed with phosphorothioated CpG ODN 1826(42-44) (Eurofins) and diluted to 1.0 mg/mL in pH 7.4 PBS. Then, an equal volume of Alhydrogel 2% (InvivoGen) was carefully added dropwise, followed by a brief period (10 min) of gentle inversion. Vaccines prepared in this manner contained 100 μg conjugate, 100 μg CpG, and 100 μL Alhydrogel, per 200 μL of complete formulation.(45)

Example 3: Synthesis of (S)-3-(3-(1-Methylpyrrolidin-2-yl)pyridin-4-yl)propan-1-amine ((−)-N4N, 6)

A. Ethyl 3-(3-((S)-1-methylpyrrolidin-2-yl)-1-pivaloyl-1,4-dihydropyridin-4-yl)propanoate (3)

A solution of ethyl 3-iodopropionate (5.93 g, 26 mmol) in THF (40 m L) was treated with zinc powder (2.04 g, 31.2 mmol). Then, copper (1) cyanide (1.79 g, 20 mmol) and lithium chloride (1.78 g, 42 mmol) in THF (40 mL) was added. Meanwhile, in a separate flask, (−)-(S)-nicotine (3.24 g, 20 mmol) in THF (80 mL) was treated with trimethylacetyl chloride (2.41 g, 20 mmol). The contents of the two flasks were mixed and stirred overnight.

The reaction mixture was cooled to 0° C., then quenched by the addition of 10% aqueous NH₄OH (150 mL). The mixture was filtered, and the filtrate was concentrated in vacuo. The remaining aqueous layer was extracted with EtOAc (3×100 mL), and the combined organic layers were washed with 10% aqueous NH₄OH (100 mL), saturated aqueous NaHCO₃ (100 mL), brine (100 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. Purification by flash chromatography (silica gel, 90:10:1 EtOAc/MeOH/NH₄OH) afforded the title compound (2.93 g, 42%) as a pale yellow semi-solid. R_(f)=0.70 (silica gel, 90:10:1 EtOAc/MeOH/NH₄OH). ¹H NMR (500 MHz, CDCl₃) δ 7.23 (s, 1H), 7.12 (d, J=8.0 Hz, 1H), 4.98 (dd, J=8.0, 5.0 Hz, 1H), 4.09 (q, J=7.3 Hz, 2H), 1.34 (s, 9H), 1.23 (t, J=7.0 Hz, 3H). LCMS (ES-API) Positive mode: 349.2 [M+H⁺] observed.

B. Ethyl (S)-3-(3-(1-methylpyrrolidin-2-yl)pyridin-4-yl)propanoate (4)

A solution of 3 (2.93 g, 8.32 mmol) in xylene (12.5 mL) at room temperature was treated with sulfur powder (0.35 g, 10.8 mmol). The resulting mixture was stirred at reflux for 90 min, and then cooled to room temperature. Purification by flash chromatography (silica gel, 90:10:0→90:10:1 EtOAc/MeOH/NH₄OH) afforded the title compound (1.09 g, 50%) as a pale yellow oil. R_(f)=0.58 (silica gel, 90:10:1 EtOAc/MeOH/NH₄OH). [α]_(D)=−115° (c 2.00, EtOH). IR (neat) ν_(max) 2969, 2939, 2777, 1730, 1592, 1178, 1158, 1042, 831 cm⁻¹H NMR (600 MHz, CDCl₃) δ 8.77 (s, 1H), 8.39 (d, J=5.1 Hz, 1H), 7.05 (d, J=5.1 Hz), 4.14 (q, J=7.1 Hz, 2H), 3.46-3.36 (m, 1H), 3.34-3.26 (m, 1H), 3.00 (t, J=7.9 Hz, 2H), 2.60 (t, J=7.9 Hz, 2H), 2.36-2.24 (m, 2H), 2.22 (s, 3H), 2.06-1.95 (m, 1H), 1.90-1.80 (m, 1H), 1.77-1.66 (m, 1H), 1.25 (t, J=7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 172.4, 149.5, 148.0, 147.3, 136.7, 123.3, 65.6, 60.8, 57.0, 40.7, 34.7, 34.6, 26.8, 22.9, 14.3. HRMS (ESI-TOF) calcd. for C₁₅H₂₂N₂O₂H⁺ [M+H⁺] 263.1754, found 263.1755.

LCMS (ES-API) Positive mode: 263.2 [M+H⁺] observed.

C. (S)-3-(3-(1-Methylpyrrolidin-2-yl)pyridin-4-yl)propanamide (5)

A solution of 4 (52 mg, 0.20 mmol) in NH₄OH (0.5 mL) was stirred at room temperature for 8 h. The solution was concentrated in vacuo. Purification by flash chromatography (silica gel, 90:10:0-80:20:2 EtOAc/MeOH/NH₄OH) afforded the title compound (35 mg, 74%) as a colorless glass. R_(f)=0.17 (silica gel, 90:10:1 EtOAc/MeOH/NH₄OH). [α]_(D)=−110° (c 1.14, EtOH). IR (neat) ν_(max) 3182, 2954, 1664, 1598, 1411, 1039, 836 cm⁻¹. ¹H NMR (600 MHz, CDCl₃) δ 8.66 (s, 1H), 8.37 (d, J=5.1 Hz, 1H), 7.06 (d, J=5.0 Hz, 1H), 5.86 (br s, 1H), 5.52 (br s, 1H), 3.34 (t, J=8.5 Hz, 1H), 3.24 (t, J=7.8 Hz, 1H), 3.12-2.96 (m, 2H), 2.52 (t, J=7.9 Hz, 2H), 2.33-2.21 (m, 2H), 2.17 (s, 3H), 2.01-1.92 (m, 1H), 1.87-1.80 (m, 1H), 1.73-1.65 (m, 1H). ¹³C NMR (150 MHz, CDCl₃) δ 174.0, 149.8, 148.1, 148.0, 136.5, 123.6, 66.2, 57.1, 40.8, 36.1, 34.4, 27.0, 23.0. HRMS (ESI-TOF) calcd. for C₁₃H₁₉N₃OH⁺ [M+H⁺]234.1601, found 234.1601. LCMS (ES-API) Positive mode: 234.2 [M+H⁺] observed.

D. (S)-3-(3-(1-Methylpyrrolidin-2-yl)pyridin-4-yl)propan-1-amine (6, (−)-N4N)

A solution of 5 (11 mg, 0.05 mmol) in toluene (0.65 mL) was treated with Red-Al (65 wt % in toluene, 64 μL, 0.21 mmol, 4.5 equiv) and stirred at room temperature for 90 min. A green reaction solution was obtained. Celite (11 mg), Darco G-60 (6 mg), and H₂O (110 μL) were added, and the mixture was filtered. Purification by preparative thin layer chromatography (0.5 mm silica gel plate, 4:1 CHCl₃/MeOH with 2% NH₄OH, major band R_(f)=0.4) afforded the title compound (5.1 mg, 49%) as a pale yellow oil. R_(f)=0.38 (silica gel, 4:1 CHCl₃/MeOH with 2% NH₄OH). [α]_(D)=−139° (c 1.29, EtOH). IR (neat) ν_(max) 3332, 3231, 2944, 2873, 2361, 1598, 1560, 1460, 1320 cm⁻¹. ¹H NMR (600 MHz, CD₃OD) δ 8.61 (s, 1H), 8.29 (d, J=5.1 Hz, 1H), 7.25 (d, J=5.1 Hz, 1H), 3.47 (t, J=8.6 Hz, 1H), 3.24 (t, J=8.4 Hz, 1H), 2.85-2.74 (m, 4H), 2.38-2.32 (m, 2H), 2.18 (s, 3H), 2.01-1.93 (m, 1H), 1.91-1.86 (m, 1H), 1.85-1.80 (m, 2H), 1.68-1.62 (m, 1H). ¹³C NMR (150 MHz, CD₃OD) δ 151.6, 149.4, 148.1, 138.4, 125.6, 66.5, 57.9, 41.5, 40.8, 35.6, 32.8, 29.9, 23.5.

HRMS (ESI-TOF) calcd. for C₁₃H₂₁N3H⁺ [M+H⁺] 220.1808, found 220.1808.

LCMS (ES-API) Positive mode: 220.2 [M+H⁺] observed.

Example 4: Preparation of Hapten-Carrier Conjugates

Following the succination procedure in Example 2(A) above, each of (−)-N4N (6) and (−)-3′-AmNic ((−)2) were separately conjugated to three proteins BSA, FliC, and TT, respectively. Hapten densities of the final hapten-carrier conjugates were approximately 30 to 40.

Immunizations and Immunoassays General.

All animal care and use was performed according to NIH guidelines and in compliance with protocols approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute. Male BALB/c mice (n=7 per group, 25-30 g) were obtained from the internal facility and assigned randomly to vaccine or saline groups. Mice were given free access to food and water during the immunization schedule, which consisted of three (100 μL) subcutaneous injections on days 0, 21, and 42. On days 28 and 49, serum samples were obtained by tail vein bleed. On day 63, animals were anesthetized, bled by cardiac puncture, and euthanized.

Example 5: Immunization

Following the procedures of Example 2(A) and 2(B), four hapten-protein conjugates ((−)-N4N-SucFliC, (−)-N4N-SucTT, (−)-3′-AmNic-SucFliC, and (−)-3′-AmNic-SucTT) were prepared and then evaluated as vaccine immunogens for eliciting anti-nicotine antibodies. Each conjugate was formulated with Alhydrogel and CpG ODN 1826 and administered subcutaneously to BALB/c mice on days 0, 21, and 42. Bleeds were collected on days 28, 49, and 63, as illustrated below:

Example 6: Enzyme-Linked Immunosorbant Assay (ELISA)

A. 3′-AmNic Conjugates

ELISA and cross-reactive ELISA were carried out using either (−)- or (+)-3′-AmNicSucBSA, prepared in a manner analogous to the TT conjugates described above. For ELISA, plasma samples were run against their respective haptens: rat plasma from the (−)-3′-AmNicSucTT group was assayed on (−)-3′-AmNicSucBSA plates, and rat plasma from the (+)-3′-AmNicSucTT group was assayed on (+)-3′-AmNicSucBSA plates.

For cross-reactive ELISA, plasma samples were run against their antipodes: rat plasma from the (−)-3′-AmNicSucTT group was assayed on (+)-3′-AmNicSucBSA plates, and rat plasma from the (+)-3′-AmNicSucTT group was assayed on (−)-3′-AmNicSucBSA plates.

The results of ELISA and cross-reactive ELISA are summarized in Table 1 below for the (−)-3′-AmNic and (.)-3′-AmNic derived antigens. By bleed 2 (day 49), titers were approximately 100,000. Furthermore, cross-reactive ELISA results demonstrate an approximately 3- to 5-fold difference in titers, showing that plasma antibodies produced in these two groups of rats possess a measurable level of enantiodifferentiation. Importantly, plasma from the (−)-3′-AmNicSucTT group has superior capacity to bind to natural (−)-nicotine displayed by (−)-3′-AmNicSucBSA.

TABLE 1 Summary of antibody titers from enzyme-linked immunosorbent assay (ELISA) Bleed 1 Bleed 2 Bleed 3 self- cross- self- cross- self- Vaccine^(a) reactive reactive reactive reactive reactive cross-reactive (−)-3′- 66,586 15,378 127,450 23,274 164,264 35,120 AmNicSucTT (+)-3′- 40,617 17,220 87,580 27,353 135,964 44,691 AmNicSucTT ^(a)formulated with CpG ODN 1826 and Alhydrogel; ELISA in duplicate (with SEM), n = 12 per group, mid-point titers

B. Specificity of Antisera for Cognate Antigen

(−)-N4N-SucBSA and (−)-3′-AmNic-SucBSA were used as coating antigens for ELISA. FIG. 3 depicts relative ELISA titers for antisera from each vaccine group, measured against the two coating antigens.

Example 7: Radioimmunoassay

Radioimmunoassay (RIA) provides a means for determining the average binding affinity and average antibody concentration for a soluble ligand. Because the ligand is soluble and free to associate/dissociate in the analysis milieu, it offers significant advantage over ELISA, in which the ligand is immobilized on the plate surface, not to mention conjugated to a carrier protein (e.g., BSA). Thus, the equilibrium environment simulated in an RIA experiment much more closely mimics that of free nicotine distributed in the blood and brain during tobacco use. It behooves researchers in the field to routinely incorporate RIA to evaluate the immunogenic efficacy of drug of abuse vaccine formulations.

Nicotine-specific plasma antibody binding affinities and antibody concentrations were determined by competitive (RIA) using an adaptation of the procedure described by Müller.(46) First, the plasma dilution that binds ˜50% of 3H-labeled nicotine was determined. Then, the affinity constant was calculated by competition with unlabeled nicotine. Because plasma samples were pooled for each vaccine group described in Example 5 above, the measured affinity constants are average affinities for each group.

As shown in FIG. 4, average binding affinity (Kdavg) and average anti-nicotine antibody concentration ([Ab]avg) were obtained, and charted in Table 2 below. By bleed 2, an approximately 4-fold difference in [Ab]avg was observed between the (−)-3′-AmNicSucTT group and the (+)-3′-AmNicSucTT group. The approximately 4-fold difference in antibody concentration observed in bleed 2 is maintained in bleed 3. It is interesting to note that ELISA/cross-reactive ELISA results showed 3- to 5-fold difference in titers; the results of these two immunoassays (ELISA and RIA) correlate with one other.

The (−)-group gave rise to superior ELISA titers and RIA antibody concentrations with a roughly 4-fold difference observed throughout. Surprisingly, affinities were higher for (−)-nicotine in the (+)-group. Scrutiny of the ELISA results and the RIA results might suggest conflicting interpretations. In particular, it seems surprising that the binding affinity for nicotine is superior (lower Kdavg) for the rats that received the (+)-vaccine. In other words, the (+)-group's plasma antibodies appear to have slightly higher binding affinity for (−)-nicotine than the (−)-group's plasma antibodies. This feature is congruent with the findings of others(47), who reported that a human anti-nicotine mAb bound (+)-nicotine with slightly higher affinity than (−)-nicotine. Incidentally, this mAb (Nicl2) was derived from another clinically evaluated nicotine vaccine, NicQβ(48), which fell short in phase 11.

The seemingly counterintuitive result for the measured Kdavg values may be rationalized by bearing in mind that nicotine (unlike cocaine and heroin) possesses greater conformational flexibility, since it consists of two heterocyclic rings joined via a single carbon-carbon bond. Given this flexibility, either enantiomer of nicotine can adopt an appropriate conformation suitable for making critical binding interactions with an antibody's binding site. For nicotine, these include the pyridyl nitrogen serving as a hydrogen-bond acceptor and the pyrrolidinium nitrogen engaging in charge-charge and/or cation-π interaction(s).

Linker attachment can also play a role in directing antihapten antibody quantity and quality. Elsewhere, it was shown that if morphine is coupled through its C-3 position to a carrier protein, codeine (3-methylmorphine) is a more effective inhibitor (than morphine) of the resultant morphine antiserum.(49) In the present study, nicotine is linked to protein carrier via the 3′-position on the pyrrolidine ring. This 3′-linkage in the (+)-3′-AmNic conjugate may impose constraints on antibody elicitation such that the measured anti-nicotine antibodies, while being of lower quantity (as anticipated), nevertheless exhibit slightly higher affinity for free (−)-nicotine than antibodies elicited by the (−)-3′-AmNic conjugate.

TABLE 2 Summary of antibody binding affinities and concentrations from radioimmunoassay Bleed 2 Bleed 3 Kdavg [Ab]avg X Kdavg [Ab]avg X Vaccine^(a) (nM) (μg/mL) (g/mol) (nM) (μg/mL) (g/mol) (−)-3′- 23.9 ± 3.2 47.5 ± 6.6 1.99 250 ± 22 118 ± 2.7 0.47 AmNicSucTT (+)-3′- 23.8 ± 3.2 12.0 ± 1.2 0.50 111 ± 9  30.4 ± 2.6  0.27 AmNicSucTT ^(a)formulated with CpG ODN 1826 and Alhydrogel; RIA in triplicate (with SEM), n = 12 per group; units for X (μg/mL/nM) were reduced to g/mol, but should not be confused with molar mass

As a means for reconciling this seeming discrepancy between average antibody affinity and average antibody concentration, we propose the use of a composite parameter, X, defined as the ratio of [Ab]avg over Kdavg for a given pool of antisera. Supposing that two ways for improving vaccine performance are to elicit higher [Ab]avg (antibody abundance) and lower Kdavg (antibody utility) values, then as improvements are made in either/or/both of these terms, the ratio (X) will become larger. Thus, for Bleed 3, (−)-group's X=0.47, while (+)-group's X=0.27. The aim is to optimize protein design for a given ligand target (“antibody efficiency”); the inverse is widely applied in medicinal chemistry: optimizing a ligand design for a given protein target (e.g., “ligand efficiency”). Hence, the ratio (X) is a means for assessing antibody efficiency and, in turn, vaccine efficacy.

The data above demonstrate the importance of chirality in vaccine design, specifically the emphasis on mimicking the natural stereochemistry of a small molecule, be it (−)-cocaine, (−)-heroin, (−)-nicotine, or any other intended target. Because nicotine as a ligand can bind in a variety of orientations within the binding site of an anti-nicotine antibody, presumably as a consequence of the conformational flexibility of nicotine itself, vaccine design as well as linker placement is more nuanced in the case of nicotine. By contrast, the additional structural constraints in cocaine or in heroin impose greater conformational rigidity, and one observes unsurprising results for relative binding affinities of antibodies for natural versus unnatural enantiomers.

Example 8: Antinociception Assay

The purpose of this example is to demonstrate the antagonism of nicotine vaccines, as described above, against nicotine by measuring the latency of mouse paw withdrawal from a hot plate.

Four test groups of mice were immunized with the four vaccines, respectively, and according to the immunization schedule as described above in Example 5. A fifth and vaccine-naïve group of mice received saline injections during the immunization schedule. All five groups of mice were then administered nicotine and then assayed on a hot plate to measure the latency in paw withdrawal from the hot plate is measured.

Antinociception for each nicotine vaccine was expressed as a percentage of maximum possible effect (“% MPE”). % MPE=(test−baseline)/(cutoff−baseline)×100, where “test” is the latency to respond after treatment; “baseline” is the latency to respond prior to treatment; and “cutoff” is the preset time at which the test was ended in the absence of a response. A baseline measure was obtained for each animal in the five groups prior to the immunization schedule above.

The results are summarized in FIG. 5, showing efficacy of the nicotine vaccines, relative to saline, in antagonizing the pain-relieving effects of nicotine in subject groups of mice.

Example 9: Hypothermia Assay

Body temperatures of mice in the five groups described above in Example 7 were measured at 10 minutes, 30 minutes, and 60 minutes following administration of nicotine. The four tested nicotine vaccines antagonized the effect of nicotine by attenuating body temperature depression, relative to the saline control group that experienced the most pronounced depression of temperature. Results are summarized in FIG. 6.

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All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 

What is claimed is:
 1. A hapten of formula (−)-N4N


2. A hapten-carrier conjugate comprising a hapten of of formula (−)-3′-AmNic

or formula (−)-N4N

wherein the hapten is covalently bonded via a linker to a carrier protein.
 3. The hapten-carrier conjugate of claim 2, wherein the hapten is formula (−)-N4N


4. An antigen for administration to a patient, comprising the hapten-carrier conjugate of claim 2 optionally in combination with one or more adjuvants.
 5. An antiserum comprising antibodies produced in response to the antigen of claim 4, wherein the antibodies have immunological affinity for (−)-nicotine.
 6. A method of treatment of nicotine or tobacco addiction or habituation in a patient suffering therefrom, comprising administering an effective amount of the antigen of claim 4 to the patient, such that antibodies are produced in the patient having affinity for (−)-nicotine.
 7. A method of treatment of nicotine or tobacco addiction or habituation in a patient suffering therefrom, comprising administering an effective amount of the antiserum of claim 5 to the patient. 